Short hairpin RNAs (shRNAs) are typically modeled after miRNA hairpin precursors and cloned into a transfer vector that is suitable for either direct transient transfection of the shRNA, or for packaging into a lentiviral particle. Lentiviral transfer vectors are ~8-10 kb in length.

+

Choosing between Lentivirus, shRNA transfer vector, or siRNA, depends on what you are seeking to accomplish. What advantage is there to create a stable knockdown versus a transient knockdown? When RNAi is performed on an easy to transfect cell line, then siRNA is a well defined, proven and effective method.When working with a primary cell, neuron, or generally hard to transfect cell, then lentiviral particles for introducing shRNA is attractive. The shRNA transfer vector alone in theory should be easy to work with insofar as culturing the cell, DNA transfection, antibiotic selection, and then collect data. However the true purpose of the lentivirus transfer vector is for packaging into lenti particles.

-

===Transient shRNA Transfection===

+

Establishing a stable knockdown phenotype is feasible with the use of lentiviral particles. Viral particles that have a VSV-G coat protein (Santa Cruz Biotechnology Inc.) will have broad tropism. In studying effects that can be measured with transient knockdown, siRNA can be a more reliable and straightforward approach. siRNA is a user friendly technique where empirical moles of duplex/# cells or total volume (molarity) combines with a minimal number of steps and peripheral controls toward achieving results.

-

Instead of chemically synthesizing the siRNAs before introducing it in the cell, the siRNAs are made directly by the cells through an expression vector that is transiently transfected into a dividng cell. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery. While transient transfection is advantageous for fast analysis of shRNA mediated effects, stable transfection ensures long-term, reproducible as well as defined shRNA effects.

+

shRNA transfer vectors are ~7 kb DNA constructs that will be more stable to handle than RNA. However there are considerable nuances in the actual design and cloning of these vectors, in the actual establishment of antibiotic resistance toward generating a stable cell, and in controlling for the specific silencing/reproducible results. shRNA has limitations due to the nature of having such a large delivery vector producing a small hairpin substrate, and over cell passages under antibiotic selective pressure (ie puro resistance may not = Pol III shRNA cassette expression).

-

===Stable shRNA Transfection===

+

==TECHNICAL SERVICE GUIDE: siRNA==

-

For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. Stable expression is achieved by integration of the gene of interest into the target cell's chromosome: Initially the shRNA of interest has to be introduced into the cell, subsequently into the nucleus, and finally it has to be integrated into chromosomal DNA.

+

Catalog # Lot #

-

Stable expression can be influenced by two factors: The transfection method used and the vector containing the shRNA of interest. The transfection method determines which cell type can be targeted for stable integration through [http://openwetware.org/wiki/Griffin:Puromycin_Selection antibiotic selection]. While many lipofection reagents transfect DNA up to a certain amount into adherent cell lines, efficient delivery of DNA into difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods and [http://en.wikipedia.org/wiki/Nucleofection nucleofection]. Nucleofection is a non-viral method of introducing DNA molecules efficiently into the nucleus of dividing cells, therefore significantly increasing the chances of chromosomal integration of the transgene. The technology was pioneered by [http://www.amaxa.com Amaxa]

Although there is still some debate as to the effectiveness of this approach, a regular shRNA transfer vector may be able to integrate into the genome of the target cell by antibiotic selection alone. The process may occur randomly by the cell's machinery itself, possibly via DNA repair and recombination enzymes. If this phenomenon does occur, integration into inactive heterochromatin may result in little or no shRNA expression, whereas integration into active euchromatin may allow for shRNA expression. However, random integration could also lead to silencing of the shRNA cassette. Several strategies have been developed to overcome the negative position effects of random integration: Site-specific, homologous and transposon-mediated integration strategies are used but require the expression of integration enzymes or additional sequences on the plasmid.

Lentiviral particles are highly efficient at infection and stable integration of the shRNA into a cell system. To obtain the lentiviral particle, the transfer vector that contains the shRNA cassette is already flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the shRNA cassette into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome. The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the shRNA into pseudovirus particles. Viral proteins which make virus shells are provided in the packaging cell line (HEK 293T), but are not in context of the LTRs and Psi-sequences and so are not packaged into virions. Thus, virus particles are produced that are replication deficient. Lentiviral particles can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.

Providing suggestions outlined in the notes below is worth considering and may bring success.

-

[[Image:lentivirus1.jpg]]

+

===Background Info===

-

Lentiviral vectors are usually created in a transient transfection system in which a cell line (HEK293T) is co-transfected with three separate plasmid expression systems. These include the transfer vector plasmid (portions of the HIV provirus & shRNA cassette), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (ENV) of a different virus. The three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell. Pseudoviral particles are then collected and must undergo titration to measure infectivity prior to experimental use.

+

*What are the experimental results?

+

*Describe how gene knockdown is measured? qPCR / Western / IF

+

*How was the RNA reconstituted?

-

===Transfer Vector Plasmid===

+

NOTE: siRNA ships lyophilized along with RNase free water with instructions to reconstitute with 330 ul of H2O to make 10 uM solution. Having the correct molarity of the solution is critical.

+

*Molarity of siRNA vialed: 10 uM ( uM/L )

+

*Volume after reconstitution: 330 uL

+

*Mass of 1 mole of siRNA: 13800 g/mol ( 21nt X 660 g/base pair)

+

*Total mols per vial: 10 um/L X 330 uL = 3.3 nm

+

*Total grams per vial: 3.3 nm X 13800 g/mol = 45.5 ug

+

*Solution concentration: 45.5 ug/ 330 uL = 0.138 ug/uL

-

The transfer vector plasmid (~8-10 kb) contains cis-acting genetic sequences necessary for the vector to infect the target cell and for transfer of the shRNA cassette, and contains restriction sites for insertion of the shRNA. The 3’ and 5’ LTRs, the original envelop proteins, and gag sequence promoter have been removed. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery.

NOTE: If the siRNA same cat# has worked in the past, and now does not work, this may suggest RNase contamination. There are ways to determine this by running 1 pmol (17 ng) siRNA in a native 2% agarose gel, however replacing the vial is a straightforward solution.

-

However, there are distinct differences between E. Coli-dependent vector-based recombination events versus point mutations. Both can influence the fidelity of the lentivector, however the implications of point mutations in the construct is much more significant. Since the labor costs associated with cloning and production of the shRNA constructs are significant, be sure to invest your time in a transfer vector that is proven in literature or contains lot specific data for adequate controls from the vendor. DO NOT assume the vector you are purchasing on paper is the one you receive in the vial.

Currently, most siRNA expression vectors are engineered to drive siRNA transcription from the polymerase III promoters U6 and H1. These promoters are particularly suited for hairpin siRNA (shRNA) expression, since they contain all of the cis-acting promoter elements upstream of the transcription initiation site and deploy a polyT transcription termination site that leads to the addition of 2-nucleotide (nt) overhangs (UU) to shRNAs, a feature that is important for siRNA function.

+

NOTE: The researcher may have an existing transfection reagent that works on their cells in other experiments (ie cDNA). Suggest to try the same reagent and measure transfection efficiency.

-

<biblio>

+

*What time point was transfection uptake of FITC-siRNA measured?

-

#Paper1 pmid=17616554

+

-

#Paper2 pmid=10684922

+

-

</biblio>

+

-

====Inducible (conditional) transfer vectors====

+

NOTE: Measuring transfection efficiency with sc-36869 will validate that liposome-dependent siRNA entry into the cells is taking place efficiently. It is important to measure transfection efficiency 5-7 hours post transfection since this is when the optimum time point where most transfection takes place. Common methods are IF or Flow cytomtetry.

*Adherent cell (grows on the surface of the plate): What is the cell confluency at time of transfection?

-

Specific inhibition of gene expression using a stably integrated siRNA (Tiscornia et al. 2003) is not suitable for silencing genes that are involved in cell growth or survival (Zhou et al. 2006). In such cases, regulating siRNA expression in mammalian cells has become essential. Having the ability to control when and how much of a particular siRNA is expressed makes it possible to study temporal- and concentration-dependent effects on a host system and allow cells to grow prior to gene silencing by “toxic” siRNAs. Furthermore, studying a host cell's response to the presence and absence of siRNA often leads to further understanding of the target gene's function.

+

*Suspension cell (ie leukocytes/lymphocytes, cells are suspended in the media) : How many cell count # used to seed the well?

-

The inducible RNAi system uses a modified form of the regulated, tetracycline-controlled gene expression system described by Gossen and Bujard (2002) in mammalian cells for the purpose of temporal silencing target genes. This system relies on two components: a tetracycline regulatory protein (TetR), which has affinity to the tetracycline operator (TetO); and a TetO-tethered pol III promoter, whose transcription activity is blocked by binding to the regulatory TetR. In the absence of tetracycline (Tet), the TetR protein binds to the TetO sequence within the promoter and acts as a potent transcription suppressor. The shRNA expression is restored when the cell culture medium is added with either Tet or doxycycline (Dox), a Tet derivative, which binds to and causes dissociation of TetR from the pol III promoter via a TetR conformation change (Zhou et al. 2006). This system allows for observation of the loss-of-function phenotypes under noninduced and induced conditions in the same isogenic cells, excluding other potential interferences.

+

NOTE: A hemocytometer (cell counter) is common for counting cells for seeding into multiwell plates (6, 12, 24 well); originally designed for performing blood cell counts. Cell density is an important parameter for knockdown. Optimum cell density will vary and typically falls between 30-80%.

+

NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%), will reveal an optimal cell density where knockdown is optimal with minimal cell death. Effective confluence can range from 30-80%.

-

<biblio>

+

===siRNA Concentration===

-

#Paper1 pmid=17616554

+

-

#Paper2 pmid=12518064

+

-

#Paper3 pmid=12552109

+

-

#Paper4 pmid=12429690

+

-

#Paper5 pmid=16787283

+

-

#Paper6 pmid=19017805

+

-

</biblio>

+

-

===Envelope Gene Plasmid===

+

*What nanomolar concentration(s) of siRNA are tested?

-

The third plasmid’s envelope gene of a different virus specifies what type of cell to target and infect. Normally HIV can infect only helper T-cells because they use their gp120 protein to bind to the CD4 receptor. However, it is possible to genetically exchange the CD4 receptor-binding protein for another envelope protein that codes for different cell types. This gives the HIV lentiviral vector a broad range of possible target cells. There a few types of heterologous envelope proteins.

+

-

There are two major points to consider when choosing which coat (pseudotype). First, because VSV-G pseudotyped virus may infect human tissue, it may not be preferable to use if one is working strictly in a mouse system. Working with human infectious agents will likely require a heightened BLS lab safety level. A second point of consideration is the limitations imposed by the ecotropic coat: it is difficult to concentrate the virus due to the inherent instability of the ecotropic coat. Virus ultracentrifugation concentration is often necessary when infecting certain cell types or when generating transgenic mice, and for this reason, many choose to use VSV-G pseudotyped virus because the particles are stable to the high g-forces of ultracentrifugation. Other [http://systembio.com/PEGIT.htm Polyethylene Glycol based concentration methods] exist that may effectively address this concern. Even with highly concentrated virus, there is a possibility that the VSV-G or ecotropic pseudotypes may still display differing cell-type specificities for infection, an important consideration when planning an experiment. For example, in the CNS, VSV-G mediates vector entry primarily in neurons, while astrocytes appear to be better infected using other envelopes.

+

NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%) & a range of siRNA concentrations (30, 60, 90 nM) will reveal an optimal convergence of cell density and concentration of siRNA where knockdown is optimal with minimal cytotoxicity (cell death).

-

There are a growing number of examples of viral coat proteins for pseudotyping the virus that influences the [http://en.wikipedia.org/wiki/Viral_tropism tropism] of infection in cell type in vitro, and tissue/organ type in vivo. For in vitro use, envelope glycoproteins and culture conditions employed should be carefully evaluated in order to address variable infectivity and phenotype awareness for each application.

+

*What time points is RNAi measured?

-

====Envelope (viral coat) proteins====

+

NOTE: 48 hours post transfection is a relevant singular point. Measuring knockdown for a few time points in the 24-72 hour window may indicate the frame when RNAi is most optimal. Titrating the siRNA concentration (30-90 nM) for the cells will indicate the best amount to see an effect.

NOTE: Primary cell cultures are first generation cells from a living organism and typically have less than 5 passage lifespan. Lenti is popular for primary cells since they are difficult to transfect. Continuous or immortalized cells have the ability to proliferate indefinitely in culture.

-

Less inflammatory with tropism for the liver. RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype.

+

-

<biblio>

+

*Is this cell type known to have tropism for VSV-G coat protein?

-

#Paper1 pmid=14722297

+

*How is transduction efficiency measured for tropism to VSV-G?

-

#Paper2 pmid=11222688

+

-

#Paper3 pmid=12186920

+

-

</biblio>

+

-

=====Semliki Forest virus (SFV; alphavirus)=====

+

*If a copGFP expressing Lentivirus was used to measure tropism, at what time point was transduction efficiency of the copGFP Control Lentiviral Particles or other reporter measured?

-

VSV-G and RRV pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments.

+

-

<biblio>

+

NOTE: 48 hours post-transduction is the time point where puromycin selection begins. This is also a good time point to evaluate transduction efficiency by measuring copGFP fluorescence inside the cells.

-

#Paper1 pmid=14722297

+

-

</biblio>

+

-

=====Sindbis virus (SINV; alphavirus)=====

+

*How was the reporter gene measured? (FCM, IF, other)

-

Sindbis virus encodes two transmembrane envelope proteins, E1 and E2. E2 is responsible for receptor binding; E1 is responsible for pH-dependent fusion. SINV when modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody.

+

-

<biblio>

+

===Multiplicity of Infection (MOI)===

-

#Paper1 pmid=14722297

+

-

#Paper2 pmid=11483746

+

-

</biblio>

+

-

=====Venezuelan equine encephalitis virus (VEEV; alphavirus)=====

-

Venezuelan equine encephalitis virus (VEEV) is a distant relative of SINV, RRV, and SFV and is a representative of the New World alphaviruses. It is an arbovirus that is normally maintained in a “silent” enzootic cycle between mosquitoes of Culex (Melanoconion) spp. and rodent reservoir hosts.

-

<biblio>

+

*X = uL of virus

-

#Paper1 pmid=15613303

+

*Y = cell count

-

</biblio>

+

-

=====Vesicular stomatitis virus G glycoprotein (VSV-G)=====

+

MOI = X* (particles/uL) /Y

-

[http://en.wikipedia.org/wiki/Vesicular_stomatitis_virus VSV] is a highly efficient particle production. Broad tropism. Less inflammatory with in vivo tropism for the liver. Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. Two important parameters of the VSV-G-pseudotyping phenomenon relating to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions.

+

-

<biblio>

+

NOTE: A hemocytometer is common for this step; originally designed for performing blood cell counts, contains a etched grid on a slide, count cells/square in 5-10 squares, then average out the number and extrapolate.

-

#Paper1 pmid=8396259

+

-

#Paper2 pmid=11689655

+

-

#Paper3 pmid=10849549

+

-

#Paper4 pmid=11482987

+

-

#Paper5 pmid=17366519

+

-

</biblio>

+

-

===Packaging Plasmid===

+

NOTE: HEK293T and other easy to transduce cells (MOI of 5-20), while neuronal cells,SHSY5Y, may require MOI of 10-50 particles/cell.

*How many [ug/ml] puromycin is added at what time point post transduction?

+

*How was optimum puromycin concentration determined?

+

NOTE: The minimum antibiotic concentration to use is the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection (normal range; 1-10 ug/ml). Add puromycin 48 hours post transduction.

+

*Western blot, IF or Quantitative RT-PCR?

-

The packaging plasmid is the backbone of the virus system. In this plasmid are found the elements required for vector packaging such as structural proteins, HIV genes (except the gene env which codes for infection of T cells, or the vector would only be able to infect these cells), and the enzymes that generate vector particles.

+

*Negative controls (scrambled hairpin virus, no virus)?

-

====gag/rev/pol====

+

NOTE: Running a parallel transduction with no virus should yield 100% cytotoxicity upon puromycin addition. Scrambled hairpin virus (sc-108080) transduction is useful to determine if any other aspect of the transduction process influences knockdown, including the presence of a non gene specific hairpin (nonspecific antisense).

Instead of chemically synthesizing the siRNAs before introducing it in the cell, the siRNAs are made directly by the cells through an expression vector that is transiently transfected into a dividng cell. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery. While transient transfection is advantageous for fast analysis of shRNA mediated effects, stable transfection ensures long-term, reproducible as well as defined shRNA effects.

-

Also contained is the human cytomegalovirus (hCMV) which is responsible for the expression of the virus proteins (gag, rev, pol) during translation. The packaging signals and their adjacent signals are removed so the parts responsible for packaging the viral DNA have been separated from the parts that activate them. Thus, the packaging sequences will not be incorporated into the viral genome and the virus will not reproduce after it has infected the host cell.

+

==Stable shRNA Transfection==

-

<biblio>

+

For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. Stable expression is achieved by integration of the gene of interest into the target cell's chromosome: Initially the shRNA of interest has to be introduced into the cell, subsequently into the nucleus, and finally it has to be integrated into chromosomal DNA.

Stable expression can be influenced by two factors: The transfection method used and the vector containing the shRNA of interest. The transfection method determines which cell type can be targeted for stable integration through [http://openwetware.org/wiki/Griffin:Puromycin_Selection antibiotic selection]. While many lipofection reagents transfect DNA up to a certain amount into adherent cell lines, efficient delivery of DNA into difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods and [http://en.wikipedia.org/wiki/Nucleofection nucleofection].

Nucleofection is a non-viral method of introducing DNA molecules into the nucleus of dividing cells, therefore significantly increasing the chances of chromosomal integration of the transgene. The technology is pioneered by [http://www.amaxa.com Amaxa]

-

Vector titration typically involves PCR or flow cytometry depending on the presence of a fluorescent protein. For example, mouse NIH 3T3 or human HT1080 cells are infected for 4 hours with limiting dilutions of vector supernate in the presence of polybrene and analyzed for expression of fluorescent protein or detection of vector or packaging sequences by PCR. The titer represents the relative infectivity of the vector as measured on the target cell of choice and is expressed as Infectious Units (IU)/mL. Different target cells, or different infection protocols, may yield different results.

+

Santa Cruz Biotechnology, Inc. does not disclose vector map information for the sh plasmids, including RE. This removes variable of a single cut RE in the empty showing up in a cloned-in sh,

-

Vector supernate can be concentrated using ultrafiltration. Recoveries (65-90%) vary depending on the envelope used and stability of the viral particle. This technique does not separate empty from functional viral particles. Therefore, at very low titers, concentration may not always yield an improved titer due to the presence of inhibitory empty viral particles.

+

The tech writing for Lonza nucleofection would suggest a linear plasmid has more efficient outcome for GFP expression , however

-

===Packaging cells===

+

*Values are so close between linear and circular DNA for the 2 and 5ug event looking at GFP expression and there is no claim that linear is necessary.

-

Human 293 cells are usually used for packaging, since they can be transfected with efficiencies in the range of 90-100%. If the cells do not transfect well in control experiments using a non-viral GFP vector, it may be worth discarding them and trying a different lot of cells. On occasion, cells can become refractive to transfection. Consequently, use low passage cells for all transfections. Although 293 cells are adherent, they can easily detach from the plate during pipetting, or even by jostling a plate that has reached confluency. For this reason, perform the transfection when the cells are approximately 30% confluent. After 36-48 hours, the cells should reach confluency and be producing the maximum amount of virus. If the cells are fed fresh media, several harvests of virus can be made after the cells reach confluency. One final note: if human cells are used for packaging a RNA hairpin that targets a human sequence, lower viral titers may be experienced if silencing of the targeted gene is detrimental to the cell or packaging process.

+

*n= 2 cell lines tested provides limited insight into the validity of the claim for such a device as this : http://www.lonzabio.com/technology.html

-

===Multiplicity of infection (MOI)===

+

*Transgene study only - there is no mention of RNAi or lenti/sh vector suitability to this tech in their nucleofection literature -

-

Multiplicity of infection (MOI) is the ratio of transfer vector transducing particles to cells. An MOI of 10 indicates that there are ten transducing units for every cell in the well. It is important to note that different cell types may require different MOIs for successful transduction and knockdown of the target gene. For instance, HEK293T cells are highly susceptible to lentiviral transduction (MOI of 5-20) while neuronal cells such as SHSY5Y often require higher MOIs of 10-50.

-

200 uL containing 10e6 particles = 50,000 particles/uL

+

===Vector dependent===

+

Although there is still some debate as to the effectiveness of this approach, a regular shRNA transfer vector may be able to integrate into the genome of the target cell by antibiotic selection alone. The process may occur randomly by the cell's machinery itself, possibly via DNA repair and recombination enzymes. If this phenomenon does occur, integration into inactive heterochromatin may result in little or no shRNA expression, whereas integration into active euchromatin may allow for shRNA expression. However, random integration could also lead to silencing of the shRNA cassette. Several strategies have been developed to overcome the negative position effects of random integration: Site-specific, homologous and transposon-mediated integration strategies are used but require the expression of integration enzymes or additional sequences on the plasmid.

-

*HEK293= 5-10 particles/cell

+

===Lentiviral particle dependent===

-

*SHSY5Y= 10-50 particles/cell

+

Lentiviral particles are highly efficient at infection and stable integration of the shRNA into a cell system. To obtain the lentiviral particle, the transfer vector that contains the shRNA cassette is already flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the shRNA cassette into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome. The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the shRNA into pseudovirus particles. Viral proteins which make virus shells are provided in the packaging cell line (HEK 293T), but are not in context of the LTRs and Psi-sequences and so are not packaged into virions. Thus, virus particles are produced that are replication deficient. Lentiviral particles can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.

-

When transducing a lentiviral construct into a cell line for the first time, a range of volume or MOI should be tested.

*96 well plate: 1.6e4 (16,000 cells); MOIs of 0.5, 1, and 5 should be used to determine the optimal transduction efficiency and knockdown for each cell line.

+

*Reproducibly transduce cell populations

-

+

*Inducible or constitutive gene knockdown

-

==shRNA Transfer Vector Promoters==

+

-

+

-

[http://en.wikipedia.org/wiki/RNA_polymerase_III RNA Polymerase III type 3 promoters] (H1, 7SK, U6) are common in vector-based expression because they are permissive expressers, they do not require any additional sequence elements, and have simple termination signals. The most abundant cellular RNAs transcribed from a type III RNA pol III promoter are the U6 small nuclear RNA (which play a crucial role in the processing of premature RNA); the 7SK RNA (a negative regulator of RNA polymerase II elongation factor TEFb); and H1 RNA (a component of RNAse P).

+

-

+

-

Unlike the majority of RNA pol III promoters, type 3 RNA Pol III promoters do not require any additional sequence elements. In addition, termination of transcription by Pol III occurs at definitive tracts of 4–5 thymidines (T4–5) (Example below), which can be inserted downstream of shRNA coding sequences in order to ensure direct termination. These 2 specific properties enable the U6, 7SK, and H1 promoters to be ideal candidates for in vivo delivery systems of siRNAs where DNA templates can undergo transcription into small RNAs with structural features resembling active siRNA/miRNA.

+

-

+

-

In summary, Pol III promoters are relatively compact, have high activity, and require a simple poly-U tract for termination.

+

-

+

-

[[Image:PolIII.jpg]]

+

-

+

-

===U6===

+

-

+

-

U6 small nuclear RNA (snRNA) is an essential component of the eukaryotic spliceosomes, is unique in that it is synthesized by RNA polymerase III, while all other U-snRNAs are synthesized by RNA polymerase II. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase II regulatory sequence motifs. Tests for both potency and adverse metabolic effects upon primary cells indicate that U6 promoter may exert toxicity relative to H1 or 7SK due in part to higher activity.

+

-

+

-

====U6 snRNA Class III gene initiation====

+

-

+

-

1. SNAPc (SNRNA Activating Protein complex) (also termed PBP and PTF) binds to the PSE (Proximal Sequence Element) centered approximately 55 base pairs upstream of the start site of transcription. This assembly is greatly stimulated by the Pol II transcription factors Oct1 and STAF that bind to an enhancer-like DSE (Distal Sequence Element) at least 200 base pairs upstream of the start site of transcription. These factors and promoter elements are shared between Pol II and Pol III transcription of snRNA genes.

+

-

+

-

2. SNAPc acts to assemble TFIIIB at a TATA box centered 26 base pairs upstream of the start site of transcription. It is the presence of a TATA box that specifies that the snRNA gene is transcribed by Pol III rather than Pol II.

+

-

+

-

3. The TFIIIB for U6 snRNA transcription contains a smaller Brf1 paralogue, Brf2. TFIIIB is the transcription factor that assembles Pol III at the start site of transcription. Sequence conservation predicts that TFIIIB containing Brf2 also plays a role in promoter opening.

+

-

+

-

===7SK===

+

-

+

-

7SK is an abundant and evolutionarily conserved small nuclear RNA discovered in the 1970s. 7SK is transcribed by RNA polymerase III from one or more genes belonging to a family of interspersed repeats in the mammalian genome (Murphy et al 1984)]. The human 7SK promoter presents a strong permissivity for the nucleotide in the +1 position and recognizes a cluster of 4 or more T residues as a termination signal. The human 7SK promoter is ideal for the production of shRNAs as it can generate high amounts of shRNAs (Czauderna et al 2003, Koper-Emde et al 2004). In a series of experiments aimed to compare the strength of the human 7SK, H1 and U6 promoters, the best silencing efficiencies of various target genes was consistently obtained with a 7SK promoter (unpublished data;[http://www.invivogen.com Invivogen]).

+

-

+

-

===H1===

+

-

+

-

The H1 promoter drives expression of a unique gene encoding H1 RNA, the RNA component of the human RNase P. The H1 RNA gene is transcribed by RNA polymerase III into a small RNA transcript. The human H1 promoter presents the characteristics of being unusually compact. All the essential elements for transcription, that is octamer, Staf, proximal sequence element and TATA motifs, lie within 100 bp of the 5‘ flanking region. This promoter is highly permissive for the nucleotide at the +1 position which is originally an A. The termination sequence consists of a stretch of 5 thymidines. The cleavage of the transcript after the termination signal occurs after the second uridine generating a 2 nt 3‘ overhang. The H1 promoter has been successfully used in different plasmids to create siRNAs.

+

-

+

-

===CMV===

+

-

+

-

CMV is a [http://en.wikipedia.org/wiki/RNA_polymerase_II RNA Polymerase II promoter]. This strong promoter is active in a broad range of cell types and performs better than most pol III promoters under long term selection. However, there are specific reports that CMV driven shRNA do not function properly certain cell types, including CD34+ hematopoietic progentior cells. [http://www.ambion.com Ambion] offers pSilencer™ 4.1-CMV plasmid vectors designed for gene silencing experiments in a broad range of cell lines (Cat# AM5775).

+

-

+

-

The CMV promoter is considered to be a stronger promoter than other common RNA pol II promoters used in mammalian expression vectors such as Simian virus-40 (SV40) and Rous sarcoma virus (RSV) (Foecking, 1986). In vivo, RNA pol II is primarily responsible for transcription of mRNA within the cell. The CMV promoter has the advantage of being highly active in a broad range of cell types, and it is suggested to not interfere with other transcription events as may be the case with the RNA pol III U6 and H1 promoters in some situations.

+

-

+

-

<biblio>

+

-

#Paper1 pmid=3023199

+

-

</biblio>

+

-

+

-

----

+

-

+

-

+

-

<biblio>

+

-

#Paper1 pmid=16844419

+

-

#Paper2 pmid=11410657

+

-

#Paper3 pmid=12552109

+

-

#Paper4 pmid=11960009

+

-

#Paper5 pmid=8336708

+

-

#Paper6 pmid=15493873

+

-

#Paper7 pmid=11972060

+

-

</biblio>

+

-

+

-

==shRNA Transfer Vector Transfection Reagents==

+

-

Transient gene silencing using RNAi is critically dependent on highly efficient delivery of the shRNA transfer vector or siRNAs into cells. The two conventional reagent types are Cationic lipid-based and Polymeric formulations. All commercial transfection reagents are proprietary formulations that are competing for market share by claiming certain advantages (ie, broad cell type compatibility, low cytotoxicity, high efficiency).

+

===Transfection Reagents===

-

===Cationic lipid===

+

*Cationic lipid

[[Image:cationliposome.jpg]]

[[Image:cationliposome.jpg]]

Line 289:

Line 177:

Cationic lipid transfection reagents are suitable for transfecting into a wide variety of dividing cell cultures. Commercial examples include: [http://en.wikipedia.org/wiki/Lipofectamine Lipofectamine / L2000], [http://www.dharmacon.com Dharmafect], [http://www.molecula.com/ iFect], and [http://www.mirusbio.com/ TransIT TKO]. Cationic lipids work by forming lipsomal vesicles that house the siRNA payload and bleb their way through the living cell membrane and into the cytoplasm. The efficiency of this process must be determined in order to have confidence in the knockdown effects. There are numerous commercial sources for transfection reagents for good reason; there are numerous cell types and lipsome structure will influence transfection efficiency in the multitude of experimental cell types that exist.

Cationic lipid transfection reagents are suitable for transfecting into a wide variety of dividing cell cultures. Commercial examples include: [http://en.wikipedia.org/wiki/Lipofectamine Lipofectamine / L2000], [http://www.dharmacon.com Dharmafect], [http://www.molecula.com/ iFect], and [http://www.mirusbio.com/ TransIT TKO]. Cationic lipids work by forming lipsomal vesicles that house the siRNA payload and bleb their way through the living cell membrane and into the cytoplasm. The efficiency of this process must be determined in order to have confidence in the knockdown effects. There are numerous commercial sources for transfection reagents for good reason; there are numerous cell types and lipsome structure will influence transfection efficiency in the multitude of experimental cell types that exist.

-

===Polymeric===

+

*Polymeric

Polymeric formulations have been developed and optimized for transfection of shRNA plasmid DNA into the nucleus of cultured eukaryotic cells by vendors such as [http://www.openbiosystems.com/ Open Biosystems]. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus (Pollard et al 1998). Polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

Polymeric formulations have been developed and optimized for transfection of shRNA plasmid DNA into the nucleus of cultured eukaryotic cells by vendors such as [http://www.openbiosystems.com/ Open Biosystems]. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus (Pollard et al 1998). Polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

-

==shRNA Transfer Vector Transient Transfection Procedure==

+

==shRNA Vector Transfection==

*In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.

*In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.

*Use 24 well plate. Each well has 1.9 mm2, good for working with 0.5 ml medium.

+

+

===Concentration Considerations:===

+

*Test 5μg/ml Polybrene

+

*Each well contains 4×104 cells. Will use Viral Particles at a quantity of 4×105 infection units. The concentration of provided viral particles was 5000 infections units per μl, so I will try 100 μl. The MOI is 10.

*Prepare a mixture of complete medium with Polybrene at a final concentration of 5 μg/ml.

+

*Remove media from plate wells and replace with 1ml of Polybrene/media mixture per well.

+

*Put the plate back to the incubator until lentiviral particles were thawed.

+

*Thaw lentiviral particles at room temperature and mix gently before use. This takes about 5 minutes.

+

*Infect cells by adding the indicated amount of shRNA lentiviral particles to the culture.

+

*Swirl the plate gently to mix and incubate overnight.

+

+

'''NOTE:''' Lentiviral particles were thawed at room temperature. As soon as the vial is thawed, Lentiviral particles were immediately added to the plate to avoid prolonged exposure of the particles to ambient temperature. Did not use ice.

*Examine GFP positive cells under microscope. Found that around 80% cells were GFP-positive, the signal was weak.

+

+

===Day 5 and on Culturing of Cells before Selection===

+

*Starting from Day 5, change to fresh medium and wait for cells to reach confluency in order to get enough cells for the selection.

+

Changed medium on day5, day8.

==shRNA Controls==

==shRNA Controls==

Line 354:

Line 278:

===Positive Controls===

===Positive Controls===

-

*Positive reporter vector or lentiviral particles; This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The GFP Control contains a gene encoding GFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection.

*Positive shRNA knockdown control; This control contains shRNA sequence that targets GFP expression. This shRNA control has been experimentally shown to reduce GFP expression. This control serves to quickly visualize knockdown in cells expressing GFP.

*Positive shRNA knockdown control; This control contains shRNA sequence that targets GFP expression. This shRNA control has been experimentally shown to reduce GFP expression. This control serves to quickly visualize knockdown in cells expressing GFP.

*Positive reporter vector or lentiviral particles; This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The GFP Control contains a gene encoding GFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection.

+

+

====copGFP====

+

The copGFP protein is a novel natural green monomeric green fluorescent protein cloned from copepod Pontellina plumata, a type of plankton. The copGFP protein is a non-toxic, non-aggregating protein with fast protein maturation, high stability at a wide range of pH (pH 4-12), and fluorescent properties that do not require any additional cofactors or substrates.

+

+

Due to its exceptional properties, copGFP is an excellent fluorescent marker that can be used instead of EGFP (the widely used Aequrea victoria GFP mutant) for monitoring delivery of lentiviral constructs into cells. The copGFP protein has a very bright fluorescence that exceeds the brightness of EGFP by approximately a third.

+

+

The copGFP protein emits green fluorescence with the following characteristics:

+

+

*emmision wavelength max – 502 nm

+

*excitation wavelength max – 482 nm

+

*quantum yield – 0.6

+

*extinction coefficient – 70,000 M-1 cm-1

+

+

When assaying cells, DO NOT fix with methanol and minimize exposure to light. PFA/Formalin fixation works.

Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.

Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.

+

+

'''DNA:'''In terms of plasmid preparation, [http://mcmanuslab.ucsf.edu/lentivirus_RNAi.html McManus Lab] has not observed a need to use E. coli cells that are highly defective for recombination. High DNA quality usually means high transfection efficiency. All DNA preparations should be performed by Cesium prep or endotoxin-free ion exchange plasmid purification methods. If poor transfection is consistently observed, it may be worth performing a additional clean-up of the DNA. The transfection protocols described here are sensitive to the amount of DNA. It is important to optimize DNA:Transfection Reagent ratios.

Lentivirus, shRNA plasmid, or siRNA?

Choosing between Lentivirus, shRNA transfer vector, or siRNA, depends on what you are seeking to accomplish. What advantage is there to create a stable knockdown versus a transient knockdown? When RNAi is performed on an easy to transfect cell line, then siRNA is a well defined, proven and effective method.When working with a primary cell, neuron, or generally hard to transfect cell, then lentiviral particles for introducing shRNA is attractive. The shRNA transfer vector alone in theory should be easy to work with insofar as culturing the cell, DNA transfection, antibiotic selection, and then collect data. However the true purpose of the lentivirus transfer vector is for packaging into lenti particles.

Establishing a stable knockdown phenotype is feasible with the use of lentiviral particles. Viral particles that have a VSV-G coat protein (Santa Cruz Biotechnology Inc.) will have broad tropism. In studying effects that can be measured with transient knockdown, siRNA can be a more reliable and straightforward approach. siRNA is a user friendly technique where empirical moles of duplex/# cells or total volume (molarity) combines with a minimal number of steps and peripheral controls toward achieving results.

shRNA transfer vectors are ~7 kb DNA constructs that will be more stable to handle than RNA. However there are considerable nuances in the actual design and cloning of these vectors, in the actual establishment of antibiotic resistance toward generating a stable cell, and in controlling for the specific silencing/reproducible results. shRNA has limitations due to the nature of having such a large delivery vector producing a small hairpin substrate, and over cell passages under antibiotic selective pressure (ie puro resistance may not = Pol III shRNA cassette expression).

Providing suggestions outlined in the notes below is worth considering and may bring success.

Background Info

What are the experimental results?

Describe how gene knockdown is measured? qPCR / Western / IF

How was the RNA reconstituted?

NOTE: siRNA ships lyophilized along with RNase free water with instructions to reconstitute with 330 ul of H2O to make 10 uM solution. Having the correct molarity of the solution is critical.

Molarity of siRNA vialed: 10 uM ( uM/L )

Volume after reconstitution: 330 uL

Mass of 1 mole of siRNA: 13800 g/mol ( 21nt X 660 g/base pair)

Total mols per vial: 10 um/L X 330 uL = 3.3 nm

Total grams per vial: 3.3 nm X 13800 g/mol = 45.5 ug

Solution concentration: 45.5 ug/ 330 uL = 0.138 ug/uL

Did this same vial or other lot of siRNA work in the past?

NOTE: If the siRNA same cat# has worked in the past, and now does not work, this may suggest RNase contamination. There are ways to determine this by running 1 pmol (17 ng) siRNA in a native 2% agarose gel, however replacing the vial is a straightforward solution.

NOTE: The researcher may have an existing transfection reagent that works on their cells in other experiments (ie cDNA). Suggest to try the same reagent and measure transfection efficiency.

What time point was transfection uptake of FITC-siRNA measured?

NOTE: Measuring transfection efficiency with sc-36869 will validate that liposome-dependent siRNA entry into the cells is taking place efficiently. It is important to measure transfection efficiency 5-7 hours post transfection since this is when the optimum time point where most transfection takes place. Common methods are IF or Flow cytomtetry.

Cell Confluency

Adherent cell (grows on the surface of the plate): What is the cell confluency at time of transfection?

Suspension cell (ie leukocytes/lymphocytes, cells are suspended in the media) : How many cell count # used to seed the well?

NOTE: A hemocytometer (cell counter) is common for counting cells for seeding into multiwell plates (6, 12, 24 well); originally designed for performing blood cell counts. Cell density is an important parameter for knockdown. Optimum cell density will vary and typically falls between 30-80%.
NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%), will reveal an optimal cell density where knockdown is optimal with minimal cell death. Effective confluence can range from 30-80%.

siRNA Concentration

What nanomolar concentration(s) of siRNA are tested?

NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%) & a range of siRNA concentrations (30, 60, 90 nM) will reveal an optimal convergence of cell density and concentration of siRNA where knockdown is optimal with minimal cytotoxicity (cell death).

What time points is RNAi measured?

NOTE: 48 hours post transfection is a relevant singular point. Measuring knockdown for a few time points in the 24-72 hour window may indicate the frame when RNAi is most optimal. Titrating the siRNA concentration (30-90 nM) for the cells will indicate the best amount to see an effect.

Measure transduction efficiency

Transduction in what cell type?

Primary cell or Continuous/immortal cell?

NOTE: Primary cell cultures are first generation cells from a living organism and typically have less than 5 passage lifespan. Lenti is popular for primary cells since they are difficult to transfect. Continuous or immortalized cells have the ability to proliferate indefinitely in culture.

Is this cell type known to have tropism for VSV-G coat protein?

How is transduction efficiency measured for tropism to VSV-G?

If a copGFP expressing Lentivirus was used to measure tropism, at what time point was transduction efficiency of the copGFP Control Lentiviral Particles or other reporter measured?

NOTE: 48 hours post-transduction is the time point where puromycin selection begins. This is also a good time point to evaluate transduction efficiency by measuring copGFP fluorescence inside the cells.

How was the reporter gene measured? (FCM, IF, other)

Multiplicity of Infection (MOI)

X = uL of virus

Y = cell count

MOI = X* (particles/uL) /Y

NOTE: A hemocytometer is common for this step; originally designed for performing blood cell counts, contains a etched grid on a slide, count cells/square in 5-10 squares, then average out the number and extrapolate.

NOTE: HEK293T and other easy to transduce cells (MOI of 5-20), while neuronal cells,SHSY5Y, may require MOI of 10-50 particles/cell.

Puromycin Selection

How many [ug/ml] puromycin is added at what time point post transduction?

How was optimum puromycin concentration determined?

NOTE: The minimum antibiotic concentration to use is the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection (normal range; 1-10 ug/ml). Add puromycin 48 hours post transduction.

Western blot, IF or Quantitative RT-PCR?

Negative controls (scrambled hairpin virus, no virus)?

NOTE: Running a parallel transduction with no virus should yield 100% cytotoxicity upon puromycin addition. Scrambled hairpin virus (sc-108080) transduction is useful to determine if any other aspect of the transduction process influences knockdown, including the presence of a non gene specific hairpin (nonspecific antisense).

Transient shRNA Transfection

Instead of chemically synthesizing the siRNAs before introducing it in the cell, the siRNAs are made directly by the cells through an expression vector that is transiently transfected into a dividng cell. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery. While transient transfection is advantageous for fast analysis of shRNA mediated effects, stable transfection ensures long-term, reproducible as well as defined shRNA effects.

Stable shRNA Transfection

For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. Stable expression is achieved by integration of the gene of interest into the target cell's chromosome: Initially the shRNA of interest has to be introduced into the cell, subsequently into the nucleus, and finally it has to be integrated into chromosomal DNA.

Stable expression can be influenced by two factors: The transfection method used and the vector containing the shRNA of interest. The transfection method determines which cell type can be targeted for stable integration through antibiotic selection. While many lipofection reagents transfect DNA up to a certain amount into adherent cell lines, efficient delivery of DNA into difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods and nucleofection.

Nucleofection

Nucleofection is a non-viral method of introducing DNA molecules into the nucleus of dividing cells, therefore significantly increasing the chances of chromosomal integration of the transgene. The technology is pioneered by Amaxa

Santa Cruz Biotechnology, Inc. does not disclose vector map information for the sh plasmids, including RE. This removes variable of a single cut RE in the empty showing up in a cloned-in sh,

The tech writing for Lonza nucleofection would suggest a linear plasmid has more efficient outcome for GFP expression , however

Values are so close between linear and circular DNA for the 2 and 5ug event looking at GFP expression and there is no claim that linear is necessary.

Transgene study only - there is no mention of RNAi or lenti/sh vector suitability to this tech in their nucleofection literature -

Vector dependent

Although there is still some debate as to the effectiveness of this approach, a regular shRNA transfer vector may be able to integrate into the genome of the target cell by antibiotic selection alone. The process may occur randomly by the cell's machinery itself, possibly via DNA repair and recombination enzymes. If this phenomenon does occur, integration into inactive heterochromatin may result in little or no shRNA expression, whereas integration into active euchromatin may allow for shRNA expression. However, random integration could also lead to silencing of the shRNA cassette. Several strategies have been developed to overcome the negative position effects of random integration: Site-specific, homologous and transposon-mediated integration strategies are used but require the expression of integration enzymes or additional sequences on the plasmid.

Lentiviral particle dependent

Lentiviral particles are highly efficient at infection and stable integration of the shRNA into a cell system. To obtain the lentiviral particle, the transfer vector that contains the shRNA cassette is already flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the shRNA cassette into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome. The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the shRNA into pseudovirus particles. Viral proteins which make virus shells are provided in the packaging cell line (HEK 293T), but are not in context of the LTRs and Psi-sequences and so are not packaged into virions. Thus, virus particles are produced that are replication deficient. Lentiviral particles can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.

Transfection Reagents

Cationic lipid

Cationic lipid transfection reagents are suitable for transfecting into a wide variety of dividing cell cultures. Commercial examples include: Lipofectamine / L2000, Dharmafect, iFect, and TransIT TKO. Cationic lipids work by forming lipsomal vesicles that house the siRNA payload and bleb their way through the living cell membrane and into the cytoplasm. The efficiency of this process must be determined in order to have confidence in the knockdown effects. There are numerous commercial sources for transfection reagents for good reason; there are numerous cell types and lipsome structure will influence transfection efficiency in the multitude of experimental cell types that exist.

Polymeric

Polymeric formulations have been developed and optimized for transfection of shRNA plasmid DNA into the nucleus of cultured eukaryotic cells by vendors such as Open Biosystems. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus (Pollard et al 1998). Polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

shRNA Vector Transfection

In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.

NOTE: This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.

NOTE: Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection.

Prepare the following solutions:

NOTE: The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined experimentally beginning with 1 μg of shRNA Plasmid DNA and between 1.0 and 6.0 μl of shRNA Plasmid Transfection Reagent as outlined below. Once the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio has been identified for a given cell type, the appropriate amount of shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent complex used per well should be tested to determine which amount provides the highest level of transfection efficiency. For example, if the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio is 1 μg:1 μl, then amounts ranging from 0.5 μg/0.5 μl to 2.0 μg/2.0 μl should be tested.

NOTE: Do not add antibiotics to the shRNA Plasmid Transfection Medium.

NOTE: Optimal results may be achieved by using siliconized microcentrifuge tubes.

NOTE: Although highly efficient in a variety of cell lines, not all shRNA Plasmid Transfection Reagents may be suitable for use with all cell lines.

Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature.

Wash the cells twice with 2 ml of shRNA Transfection Medium. Aspirate the medium and proceed immediately to the next step. NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency.

NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.

Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.

Aspirate the medium and replace with fresh 1x normal growth medium.

Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the step above.

NOTE: Controls should always be included in shRNA experiments. Control shRNAs are available as 20 μg. Each encode a scrambled shRNA sequence that will not lead to the specific degradation of any known cellular mRNA.

NOTE: For Western blot analysis prepare cell lysate as follows: Wash cells once with PBS. Lyse cells in 300 μl 1x Electrophoresis Sample Buffer (sc-24945) by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary.

Lentivrus Infection

Materials:

Use 24 well plate. Each well has 1.9 mm2, good for working with 0.5 ml medium.

Concentration Considerations:

Test 5μg/ml Polybrene

Each well contains 4×104 cells. Will use Viral Particles at a quantity of 4×105 infection units. The concentration of provided viral particles was 5000 infections units per μl, so I will try 100 μl. The MOI is 10.

Day 2 Lentiviral Infection

Prepare a mixture of complete medium with Polybrene at a final concentration of 5 μg/ml.

Remove media from plate wells and replace with 1ml of Polybrene/media mixture per well.

Put the plate back to the incubator until lentiviral particles were thawed.

Thaw lentiviral particles at room temperature and mix gently before use. This takes about 5 minutes.

Infect cells by adding the indicated amount of shRNA lentiviral particles to the culture.

Swirl the plate gently to mix and incubate overnight.

NOTE: Lentiviral particles were thawed at room temperature. As soon as the vial is thawed, Lentiviral particles were immediately added to the plate to avoid prolonged exposure of the particles to ambient temperature. Did not use ice.

Day 3 Change Medium

Examine GFP positive cells under microscope. Found that around 80% cells were GFP-positive, the signal was weak.

Day 5 and on Culturing of Cells before Selection

Starting from Day 5, change to fresh medium and wait for cells to reach confluency in order to get enough cells for the selection.

Changed medium on day5, day8.

shRNA Controls

Negative Controls

Untreated Cells. Untreated cells will provide a reference point for comparing all other samples.

Empty construct, containing no shRNA insert; The empty viral particles or DNA are a useful negative control that will not activate the RNAi pathway because it does not contain an shRNA insert. It will allow for observation of cellular effects of the transduction/transfection process. Cells transduced/transfected with the empty control provide a useful reference point for comparing specific knockdown.

Non-targeting shRNA; This non-targeting shRNA is a useful negative control that will activate RISC and the RNAi pathway, but does not target any human or mouse genes. The short hairpin sequence cotnains 5 base pair mismatches to any known human or mouse gene. This allows for examination of the effects of shRNA transduction/transfection on gene expression. Cells transduced/transfected with the non-target shRNA will also provide useful reference for interpretation of knockdown.

Positive Controls

Positive shRNA knockdown control; This control contains shRNA sequence that targets GFP expression. This shRNA control has been experimentally shown to reduce GFP expression. This control serves to quickly visualize knockdown in cells expressing GFP.

Positive reporter vector or lentiviral particles; This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The GFP Control contains a gene encoding GFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection.

copGFP

The copGFP protein is a novel natural green monomeric green fluorescent protein cloned from copepod Pontellina plumata, a type of plankton. The copGFP protein is a non-toxic, non-aggregating protein with fast protein maturation, high stability at a wide range of pH (pH 4-12), and fluorescent properties that do not require any additional cofactors or substrates.

Due to its exceptional properties, copGFP is an excellent fluorescent marker that can be used instead of EGFP (the widely used Aequrea victoria GFP mutant) for monitoring delivery of lentiviral constructs into cells. The copGFP protein has a very bright fluorescence that exceeds the brightness of EGFP by approximately a third.

The copGFP protein emits green fluorescence with the following characteristics:

emmision wavelength max – 502 nm

excitation wavelength max – 482 nm

quantum yield – 0.6

extinction coefficient – 70,000 M-1 cm-1

When assaying cells, DO NOT fix with methanol and minimize exposure to light. PFA/Formalin fixation works.

Factors Influencing Successful Transfection

Concentration and purity of nucleic acids

Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.

DNA:In terms of plasmid preparation, McManus Lab has not observed a need to use E. coli cells that are highly defective for recombination. High DNA quality usually means high transfection efficiency. All DNA preparations should be performed by Cesium prep or endotoxin-free ion exchange plasmid purification methods. If poor transfection is consistently observed, it may be worth performing a additional clean-up of the DNA. The transfection protocols described here are sensitive to the amount of DNA. It is important to optimize DNA:Transfection Reagent ratios.

Transfection in serum-free media

The highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is observed.

No antibiotics in transfection medium

The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.

High protein expression levels

Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.

Cell history, density, and passage number

It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.